Tyrosine kinase activity is a crucial component of cellular signaling cascades. Precise regulatory control over kinase activity must be maintained during signaling as evidenced by numerous human diseases that arise upon dysregulation of protein kinases. This project is aimed at generating a molecular level understanding of how tyrosine kinase activity is regulated during T cell signaling. The current renewal application continues to focus on the Tec family immunological tyrosine kinase Interleukin-2 tyrosine kinase, Itk. Our work over the last period has generated preliminary data that support mechanistic models for Itk regulation. Specifically, we have identified Itk dimerization as a switch for turning Itk catalytic activity 'off'; we have discovered a specific docking interaction between Itk and its substrates that ensures fidelity in target phosphorylation; and we have data that suggest that the peptidyl prolyl isomerase, cyclophilin A, controls Itk activity by preventing substrate docking. Our studies on Itk and related Tec family members have also extended our understanding of substrate recognition for the well studied Src family of tyrosine kinases. The Src kinase, Lck, activates Itk following TCR engagement by phosphorylating a specific tyrosine in the Itk kinase domain and we have now identified that this reaction occurs via recognition of a remote substrate docking site. The aims proposed in this application will pursue detailed structural studies of all of the protein regulatory complexes described. The molecular level knowledge that will emerge from this work will provide a better understanding of T cell signaling and the means to target specific interactions for therapeutic uses. PUBLIC HEALTH RELEVANCE: This proposal aims to understand specific protein interaction leading to activation of tyrosine kinases in the immune response. The public health relevance of the project relates to developing new ways to either limit or enhance the immune response in the face of autoimmunity, immunosuppression or immunological diseases. Kinases in particular are prime therapeutic targets since they control much of immune cell signaling and genetic defects in specific kinases are linked to specific human diseases.